The Molecular Imaging Branch (MIB) aims to exploit positron emission tomography (PET) as a radiotracer imaging technique for investigating neuropsychiatric disorders, such as autism, depression, addiction, schizophrenia, and Alzheimer's Disease (AD). Fundamental to this mission is the development of novel radioactive probes (radiotracers) that can be used with PET to measure changes in low level proteins in the brains of living human subjects where these proteins are suspected to have critical involvement in the progression of neuropsychiatric disorders. Such proteins include some neuroreceptors, transporters, enzymes, and plaques. PET is uniquely powerful and sensitive when coupled with the use of biochemically-specific PET radiotracers. The chemical development of new radiotracers is the key to exploiting the full potential of PET in neuropsychiatric research. However, a successful PET radiotracer must satisfy a wide range of difficult-to-satisfy chemical, biochemical and pharmacological criteria. Consequently, PET radiotracer development is highly challenging. In fact, our research has some parallels with drug discovery in that it entails high effort and heavy risk, but can reap rich biomedical rewards. Even now, the number of potentially interesting imaging targets (brain proteins) far exceeds the range of currently available and useful radiotracers. Within MIB, the PET Radiopharmaceutical Sciences Section (PRSS) places a concerted effort on all chemical and radiochemical aspects of PET radiotracer discovery. Our laboratories are equipped for medicinal chemistry and automated radiochemistry with positron-emitting carbon-11 (t1/2 = 20 min) and fluorine-18 (t1/2 = 110 min). These two very short-lived radioisotopes are available to us daily from the adjacent cyclotrons of the NIH Clinical Center (Chief: Dr. P. Herscovitch). Our section interacts seamlessly with the Section on PET Neuroimaging Sciences in our branch (Chief: Dr. R.B. Innis) for early evaluation of potential radiotracers in biological models and in animals. Subsequent PET research in human subjects is also performed in collaboration with the Imaging Section under Food and Drug Administration oversight through 'exploratory' or 'full' Investigational New Drug applications. In the period covered by this report, we worked on developing PET radiotracers for several targets. These include TSPO; many receptors (glutamate(NMDA),serotonin subtype 1B (5-HT1B), histamine subtype-3 (H3)); enzymes (COX-1, COX-2, PDE1 and PDE4 subtypes, cPLA2, OGA); and tau fibril deposits. One radiotracer that we earlier developed for TSPO imaging (C-11PBR28) has been applied by many imaging centers to investigate brain inflammatory conditions in response to various neurological insults (e.g., stroke, epilepsy and neurodegeneration). An unexpected finding is that healthy human subjects, because of a genetic difference, carry either one or both of two distinct forms of TSPO and that these interact differently with C-11PBR28, complicating the analysis of PET studies. Consequently, we sought to develop genetically-insensitive TSPO radiotracers. We explored new chemotypes with potential to provide superior PET radiotracers for TSPO. One of our new radiotracers, C-11ER176, appeared promising for avoiding genotype sensitivity from study in animals and in human tissue in vitro. Although C-11ER176 does not show the expected genotype insensitivity in living humans, it does turn out to be perhaps the highest performing TSPO radiotracer yet known. We and other imaging centers now plan to use this radiotracer for clinical studies in human subjects. In addition, we are developing radiotracers for other targets relevant to the study of neuroinflammation, such as the cyclooxygenase (COX) subtype 1 and subtype 2 enzymes. Very promising radiotracers have been identified, and two of these are to be advanced for evaluation in human subjects. These radiotracers may provide more biochemical and cellular specificity for investigation of neuroinflammation. In this regard, we have started to explore the development of radiotracers for other enzymes implicated in neuroinflammation, such as cPLA2. mGlu5 receptor radiotracers have interest for the study of addiction, as well as other disorders, notably Fragile X syndrome associated with autism spectrum disorder, and schizophrenia. We reported our comparison of two new high-performing mGlu5 radiotracers (C-11SP203 and C-11FPEB) in human subjects and our recently developed radiotracer (F-18FIMX) for a very related receptor target, namely mGlu1. Studies in human show this radiotracer performs exceptionally well. NMDA is another protein acted upon by the important neurotransmitter, glutamate, for which we continue developing radiotracers. In collaboration with Prof. Diaz-Arrastia and colleagues (Uniformed Services University of the Health Sciences), we have been developing radiotracers for imaging the accumulation of brain neurofibrillary tangles (tau protein), which may underlie the development of neurodegenerative disorders, such as AD and traumatic brain injury. We have identified three distinct radiotracer binding sites on tau fibrils and have discovered several compounds that might serve as leads to PET radiotracers for tau imaging. Ligand-based virtual screening has also been explored as a tool for PET radiotracer discovery. PET radiotracers can provide important quantitative information on experimental therapeutics for neuropsychiatric disorders, such as ability to cross the blood-brain-barrier and to engage with a target protein. In collaborations with academia and Pharma, we are developing several radiotracers for this purpose. Some of these radiotracers are targeted at proteins that have not previously been imaged in living human brain, and that may have eventual clinical research utility. These proteins include subtypes of phosphodiesterase (especially with regard to depression), and the enzyme OGA (especially with regard to dementia). We are currently evaluating a very promising radiotracer for OGA in human subjects. The development of radiotracers for phosphodiesterase subtypes shows early promise. We are advancing our methodology for improved radiotracer development. New labeling agents have been developed for C-11 chemistry, notably C-11fluoroform which opens up wide chemical space for potential radiotracer development. We have also succeeded in developing a method for F-18fluoroform synthesis at a higher molar activity than hitherto possible, again expanding possibilities for new radiotracer development. New radiolabeling chemistries have been developed to utilize C-11fluoroform or F-18fluoroforms. Sensitive mass spectrometry has been developed to measure radiotracer concentration in blood following intravenous administration, as is required to analyze PET experiments. LC-MS/MS has the potential to avoid the demanding logistics associated with measuring fast-decaying radioactivity. Productive collaborations continue with external academic chemistry and medicinal chemistry laboratories, nationally and internationally, and with pharmaceutical companies. Productive collaborations also exist with other centers working with PET and its associated radiochemistry and radiotracer development. The laboratory is active in training new scientists for this field at graduate and postdoctoral level. We produce some useful radiotracers that have been developed elsewhere for PET investigations in animal or human subjects e.g., C-11rolipram (for PDE4 enzyme imaging), and C-11clozapine for investigation of DREAAD technology. Each PET experiment with any radiotracer requires a radiosynthesis of the radiotracer on the same day, and hence radiotracer production is a regular activity.